Evidence indicates that steroidogenic cells are capable of interiorizing lipoprotein-delivered cholesterol by a non-endocytic or 'selective' pathway. In this pathway cells preferentially take in lipoprotein-supplied cholesteryl esters (CE), relative to the uptake of lipoprotein apoproteins. The selective pathway has been shown to occur in a variety of human and animal cells in culture, but it is especially important in the ovary or adrenal where it delivers CE in bulk to cells which require cholesterol as a precursor for steroid hormone production. Thus, in our zeal to examine ways to reduce circulating cholesterol levels in individuals it is important to remember that some cells require large amounts of exogenous-provided cholesterol for their specific functions. Despite extensive use of the 'selective' pathway by cells, we are just now beginning to understand how the CE of this pathway is internalized and transported intracellularly. Recent acquisition of two major technical advances in the field have provided the means of obtaining important basic information on these questions. First, we have been able to trace the uptake and intracellular transport of a fluorescently labeled CE(BODIPY(R)-CE prepared as a component of reconstituted apo E free high density lipoprotein, hHDL3) in both intact and semi-intact rat ovarian granulosa cells in culture. The fluorescently tagged ligand is hydrolyzable by acid compartments (e.g. lysosomes) of the cell (but not by neutral components of the 'selective' pathway) and as such, has provided us with a powerful tool by which we can specifically trace early stages of this pathway. The present application outlines a variety of steps using this probe by which we can morphologically and biochemically determine the role of membranes, specific cell organelles, cytosolic factors, cytoskeletal components and energy to 'selective' pathway function, as well as a means of identifying sequential steps in the pathway, signaling events, etc. Of special interest also is the possibility of using genetically altered animals to isolate the 'selective' pathway (from other cholesterol uptake pathways) of steroidogenic cells. Knockout mice (missing the B/E receptor) or double knockout mice (missing both the B/E receptor and lipoproteins containing apoE) are available and are currently being tested by us to establish their 'selective' pathway characteristics. These special mice with their single cholesterol pathway can confirm earlier observations regarding the 'selective' pathway, but more importantly can identify later intracellular events in the selective pathway using a fluorescent CE marker (NBD-CE) which is hydrolyzable under neutral cell conditions, and releases NBD-labeled free cholesterol which can be subsequently visualized and traced.